Project Summary Nervous system development, growth, and regeneration depend on neural stem cell (NSC) homeostasis, a state of delicate equilibrium between NSC self-renewal, differentiation, and survival. Defects in NSC homeostasis underlie broad neurodevelopmental, psychiatric, and neurodegenerative disorders. The molecular and cellular mechanisms underlying the control of NSC homeostasis remain poorly understood. We propose to elucidate the basic mechanisms underlying the genetic control of NSC homeostasis, using Drosophila larval brain type II neuroblasts (NBs) as a model. Drosophila NBs have been instrumental in discovering signaling molecules and cellular mechanisms that are centrally involved in NSC homeostasis. Similar to mammalian NSCs in lineage hierarchy, the type II NB lineages in the Drosophila larval brain contain transit-amplifying intermediate progenitors (IPs), which can generate a vast number of differentiated progenies. Notch signaling is critical for maintaining the homeostasis of type II NB lineages. Inhibition of Notch signaling results in NB not being properly maintained, whereas Notch hyperactivation causes ectopic NB formation and brain tumorigenesis. Notch signaling also regulates the homeostasis of mammalian NSCs, with deregulated N signaling having been linked to brain cancer. The molecular mechanisms by which N signaling regulates NSC homeostasis, however, are not well understood. Previous studies of N signaling in NSCs have focused heavily on canonical N signaling mediated by Suppressor of Hairless [Su(H)]- related transcription factors. However, our studies in the previous funding period have found that a novel non- canonical N signaling pathway operating in the cytosol is also critically involved. Components of this non-canonical N signaling pathway include mitochondrial PTEN-induced kinase 1 (PINK1), mechanistic target of rapamycin complex-2 (mTORC2), and mTORC2 substrate AKT. Clinical significance of this non-canonical N signaling pathway is underscored by our observation that tumor-initiating cancer stem cell (CSC)-like cells in both Drosophila brain tumor models and human GBM samples are particularly sensitive to perturbation of this pathway. Moreover, we found that this non-canonical pathway exerts translational control over mitochondrial oxidative phosphorylation related mRNAs. The goal of this proposal is to move away from the status quo of transcriptional regulation of NSCs by focusing on the newly discovered translational control mechanism in N signaling. Our central hypothesis is that non-canonical N signaling regulates NSC homeostasis through co-translational quality control of mitochondrial mRNAs, thus modulating mitochondrial proteome and function. To test this hypothesis, we propose two Specific Aims. Aim 1 will elucidate how the co-translational quality control pathway mediates the effect of Notch on NSC homeostasis. Aim 2 will dissect the molecular mechanism by which Notch regulates the co-translational quality control process. Upon successful completion of these Aims, we will have generated new mechanistic insights into the control of NSC homeostasis by Notch. We anticipate that this will open up entirely new directions for studying the fundamental roles of Notch in NSC biology, cancer biology, and adult brain function.